Rabbit Blastocysts Research Articles

Isolation and characterization of the specific peptides from rabbit blastocyst at peri-implantation stage with emphasis on their physiological significance relevant to the mechanism of implantation.

The secretory materials from the uterine flushings of the pregnant, non-pregnant and pseudopregnant rabbits were analysed by the method of SDS-polyacrylamide gel electrophoresis. The results demonstrated that there are three specific peptides in the pregnant uterine flushings with molecular weights of 3000, 4500 and 6000. No correspondent peptides were found in non-pregnant and pseudopregnant uterine flushings. The peptides were also isolated and identified from the blastocystic fluid. At least 4 peaks of small peptides were shown by high pressure liquid chromatography. The range of their isoelectrophoretic points is between pH 4.0-5.5 Concavalin A (Con A) affinitive electrophoresis indicated that they are glycopeptides in nature. The LH-RH A is able to exert inhibitory effects on the synthesis of these peptides. Their possible physiological function has been discussed.

Mineralocorticoid concentrations in unstressed female rabbits and embryonic sodium transport.

Plasma aldosterone, corticosterone, and cortisol were measured during the first week of pseudopregnancy or pregnancy in New Zealand White rabbits to determine whether any sustained elevations of adrenal steroids occur. There were no pregnancy-specific alterations in circulating adrenal steroid concentrations during the preimplantation stages of embryonic development. Elevation of plasma aldosterone in vivo did not induce amiloride-sensitive Na+ transport across the embryonic trophectoderm. It therefore seems unlikely that an increase in maternal adrenal steroid concentrations is necessary for the development of amiloride-sensitive Na+ transport in rabbit blastocysts. Sodium efflux from Day 6 post coitum (p.c.) blastocysts was lower than Na+ influx. By day 7 p.c. Na+ efflux was equivalent in magnitude to the component of Na+ influx not inhibited by amiloride. This suggests that between Days 6 and 7 p.c. the amiloride-sensitive component of Na+ influx becomes essential for blastocyst expansion.

Metallothionein gene regulation in the preimplantation rabbit blastocyst.

Expression of metallothionein (MT) genes in the preimplantation rabbit blastocyst was analysed by determination of the levels of MT mRNA and relative rates of MT synthesis. MT was found to be constitutively expressed at low levels in the blastocyst. Exposure of the day-6 blastocyst to zinc ions in vitro rapidly increased the level of MT gene expression in a dose-dependent manner, with a ten-fold induction in the relative rate of synthesis at 400 microM-Zn2+. Ion-exchange chromatography of pulse-labelled blastocyst protein showed that the relative rates of synthesis of both MT-I and MT-II were markedly increased following zinc treatment, with MT-I being the predominant isometallothionein. Zinc induction of MT synthesis in the blastocyst was also detected on day 4 of gestation just after the morula-to-blastocyst transition. In contrast to the zinc effects on MT, in vitro exposure to 10 microM-Cd2+ resulted in a large induction of MT mRNA but only a modest increase in the relative rate of MT synthesis. Cadmium was found to be toxic to the day-6 blastocyst, and 10 microM-Cd2+ induced an acute stress response as indicated by a dramatic induction of heat-shock protein (HSP-70) gene expression.

Equine zona pellucida and capsule: some physicochemical and antigenic properties.

The capsule which surrounds the pre-attachment equine embryo has been compared with the zona pellucida (zp) that it replaces, as well as with the rabbit blastocyst coverings, by means of physicochemical and immunological methods. Trypsin solution at pH varying between 7.5 and 9.0 completely solubilized the capsule, as did Na borohydride. However, solutions of pH 2.0 or 12.0, urea, high temperature (65 degrees C, 60 min or 80 degrees C, 30 min), mercaptoethanol and dithiothreitol were able to solubilize the zp but not the capsule at the concentrations used. Indirect immunofluorescence on cryostat sections and whole mounts of fresh or frozen-thawed material showed that 1) common antigens are shared by equine, porcine and bovine zp; 2) day 7 to day 15.5 capsule reacted with anti-capsule-serum but not with anti-zp-serum except for a few patches on the surface of the capsule; 3) anti-capsule-serum, but not anti-zp-serum, reacted with the capsular material recovered along with a broken day 27.5 conceptus; 4) anti-capsule-serum does not react with rabbit blastocyst coverings; 5) anti-capsule antibodies can be absorbed from the anti-capsule-serum by uterine proteins from either pregnant or non-pregnant mares; and 6) the capsule does not contain mouse laminin-like material.

Quantitative aspects of protein synthesis in non-cultured and cultured rabbit blastocysts.

Day 4 rabbit blastocysts were cultured in Ham's F-10 medium supplemented either with homologous serum or uterine flushings. Development was assessed by leucine and methionine incorporation at 24 h and 48 h, respectively, after initiation of culture and compared with day 4 and day 5 non-cultured controls. After 24 h in culture, incorporation data were similar for both media groups. After 48 h, a significantly higher protein synthesis activity was found in blastocysts which had access to uterine secretions in vitro. However, incorporation levels of day 5 controls were not reached. Modes of incorporation of leucine and methionine revealed to be very similar in all experimental groups but ranged on a different level. Leucine was incorporated on a 5.5-fold higher level than methionine. We conclude that (i) supplementation of culture media with uterine secretions is beneficial for blastocyst development, and (ii) further studies should focus on non-serum components within the uterine fluid and their significance for embryonic development.

Radioiodinated surface proteins of rabbit trophectoderm cells.

We have previously used surface iodination to discriminate between the protein patterns of epithelial cell surfaces in uteri of rabbits receptive (Day 6.5) or nonreceptive (Day 4) to nidation (Ricketts et al. 1984). In this paper, we describe application of the same technique to the trophoblastic surface of rabbit blastocysts collected on the same days of pregnancy. Analysis of labelled proteins by polyacrylamide-gel electrophoresis under denaturing conditions did not reveal qualitative differences between the two days of pregnancy. Scanning densitometry was used to quantitate the area under each protein peak on an autoradiogram; these areas were used as variables in statistical analysis of the protein pattern of individual animals. Quantitative differences between the protein patterns of the two surfaces were detected by canonical variate analysis of the pattern of relative areas of labelled protein peaks. In proteins separated on 7.5% gels, this statistical analysis correctly assigned blastocysts from 8 out of 10 animals to one of two groups according to day of pregnancy. The discrimination was not statistically significant, however, in protein patterns on 12.5% gels, used to give better separation in the lower range of molecular weights. The same analysis in the uterus unequivocally separated the surface iodination patterns from these same days of pregnancy. Thus the changes detected by surface iodination appear to be less pronounced on the trophectoderm than on the uterine epithelium in relation to the time of ovoimplantation.

Neutral amino acid influx in developing rabbit blastocysts

The influx of the neutral amino acids glycine, aminoisobutyric acid (AIB), and leucine into rabbit blastocysts was measured. In day 6 postcoitus (pc) embryos, glycine influx was Na+ independent, whereas AIB and leucine influx involved both Na+-dependent and independent components. From days 5 to 7 pc, the leucine and AIB influx remained constant, although the Na+-dependent fraction decreased and the Na+-independent fraction increased with age. None of the Na+-independent influx was inhibited by methylaminoisobutyric acid (MeAIB), an amino acid analogue specific for the system A of neutral amino acid uptake. In addition, MeAIB influx was Na+ independent, implying that system A is not involved in leucine or AIB uptake. All Na+-dependent influx is thus considered to occur via system ASC. System L contributed only to the influx of leucine at days 6 and 7 pc, as measured by inhibition of Na+-independent influx by 2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic acid.

Possible role of prostaglandin F in blastocyst implantation

The synthesis and release of Prostaglandin F (PGF) by the rabbit blastocyst and endometrium were investigated on Day 6 and 7, using radioimmunoassay, autoradiography and conversion experiments. The following results were obtained: 1. 1) The content of PGF in the blastocyst increased significantly (P < 0.01) form Day 6 and 7. 2. 2) The content of PGF in the endometrium was significantly higher (P < 0.05) on Day 7 implantation sites compared to the other areas. 3. 3) The in vitro synthesis and release of PGF by Day 6 balstocysts sharply increased after one and two hours of culture, respectively. Thereafter both values declined with time. 4. 4) The in vitro synthesis and release of PGF by Day 6 endometria increased continuously with time. 5. 5) 14C-arachidonic acid ( 14C-AA) was incorporated into Day 6 blastocysts in vitro and converted to PGF 2α. These results suggest that both the endometrium and the blastocyst are the sources of the PGs involved in implantation, and that PGF derived from the blastocysts may act as the trigger of implantation.

Direct effects of nicotine on rabbit preimplantation embryos

The effects of various concentrations of nicotine on the in vitro development of 1-cell rabbit preimplantation embryos and on the DNA-synthesis of 4-day-old rabbit blastocysts are investigated. Exposure of rabbit preimplantation embryos to concentrations of nicotine higher than 1 × 10 −3 M results in a marked decrease in the invitro development and in DNA-synthesis. Concentrations of nicotine below 1 × 10 −3 M have no effect on these early embryos. It can be concluded that the concentrations of nicotine which exert a direct embryotoxic effect are higher than the concentrations that may be expected in the blood circulation of humans considered to be “normal smokers”.

Implantation of the rabbit blastocyst: Sequential changes in estradiol and progesterone and their receptors

To determine the temporal relationship of the endocrine events involved in rabbit blastocyst implantation, (1) progesterone (P) and estradiol (E) in plasma and uterine flushings, (2) P and E in the cytosol of endometrium and myometrium, and (3) P receptors (PR) and E receptors (ER) in endometrium and myometrium were examined from days 0 through 7 post coitum by radioimmunoassay and radioreceptorassay. Plasma P levels increased significantly from 0.3 +/- 0.06 to 76 +/- 9.4 ng/ml (p = less than 0.005) 2 hours after mating (day 0), declined to 10.0 +/- 1.2 ng/ml by day 6, and increased to 12.0 +/- 1.2 ng/ml by day 7. Cytosol P levels of endometrium and myometrium increased from 3.2 +/- 0.2 to 22.8 +/- 2.2 ng/gm and 2.1 +/- 0.4 to 14.1 +/- 2.1, respectively, from days 1 to 6. On day 7, cytosol P levels of the embryonic segment (8.2 +/- 0.5 ng/gm) were lower than those of the interembryonic segment (12.4 +/- 1.8 ng/gm). P levels in uterine flushings increased significantly from 0.05 +/- 0.01 ng/ml on day 0 to 19.9 +/- 0.7 ng/ml on day 7 (p = less than 0.001). In contrast, E levels in plasma, uterine flushings, and cytosol of uterine tissues showed no significant change from days 1 through 7, but the levels from 6 hours after mating were significantly higher than those before mating (p = less than 0.05). ER in the cytosol and nucleus of all uterine regions increased significantly after mating but decreased after implantation. In contrast, PR in the cytosol and nucleus of all uterine regions showed no significant change on days 0 to 6 post coitum. After implantation, the embryonic segment had significantly higher PR in the nucleus than that in the cytosol (13.0 +/- 1.5 versus 8.0 +/- 0.5 fmol/micrograms), whereas the interembryonic segment had significantly higher PR in the cytosol than that in the nucleus (12.0 +/- 0.5 versus 9.0 +/- 0.5 fmol/micrograms DNA). These findings showed that changes in P in the plasma and uterine flushings preceded those in uterine tissues before implantation. The changes in uterine tissue ER and PR after mating but before and after implantation suggest that the putative nidatory sites are prepared for implantation but undergo endocrine changes to protect the conceptus immediately after implantation.

Effects of intrauterine instillation of antiserum to hCG during early pregnancy in mice.

Human chorionic gonadotrophin (hCG)-like activity has been reported in mouse and rabbit blastocysts. The presence of this hCG-like activity seems to be essential for nidation of the pre-implantation embryo. Binding sites for hCG were localised on day 4 mouse embryos by immunohistochemical techniques. Presence of hCG-like activity was confirmed by cytotoxicity test. The number of implantation sites was significantly reduced on day 8 of pregnancy. Prior treatment to day 4 mouse embryos with hCG antiserum, for 1 h in utero or in vitro and their subsequent transfer to uteri of synchronised pseudopregnant mice resulted in impaired implantation of embryos, compared to controls treated with normal rabbit serum (NRS). These results suggest that hCG-like activity present on the pre-implantation embryo may have a significant role in implantation of the embryo.

Rabbit blastocysts accumulate [3H]prostaglandins in vitro.

Rabbit blastocysts obtained on days 5, 6, and 6.8 of pregnancy were incubated in vitro in Tyrode's buffer with 3H-labeled prostaglandins (PGs). Accumulation of PGs was studied, using Whatman GF/F filters to separate bound and free ligands. The uptake and efflux of [3H]PGs were studied as a function of PG type, incubation time, temperature, and effect of metabolic inhibitors as well as age and number of blastocysts. Blastocysts of the same age accumulated approximately the same amount of [3H]PGE2 and [3H]PGF2 alpha from their environment; however, there was no apparent saturation over a PG concentration range of 1-1000 nM. Both the uptake and efflux of PG were age dependent, with older blastocysts accumulating more PGs. Approximately 90% of the [3H]PGs appear to be transported into the blastocoelic fluid, with little PG remaining in the blastomeres. PG accumulation was relatively insensitive to azide, ouabain, cyanide, or bromcresol green, but was affected by incubation at 0 C or the addition of indomethacin (10 micrograms/ml). No catabolism of the accumulated PGs was observed. The release of PGE2 in general did not differ from that of PGF2 alpha, except on day 6.8 of pregnancy when PGE2 was released more rapidly than on day 6. We conclude that rabbit blastocysts can accumulate PGs from their environment, which may imply a storage potential in the blastocyst and release before implantation.

Physiological aspects of blastocyst uterine interaction

An interaction between the blastocyst and the uterus is essential for establishment of pregnancy. Because maternal estrogen is not an absolute requirement, estrogen of embryonic origin has been implicated in this process in the pig and the rabbit. Furthermore, estrogen forming capacity has been documented in the blastocyst of these species. However, while the complete machinery for steroid synthesis in the pig balstocyst has been demonstrated, the issue is still unresolved for the rabbit blastocyst. In the present communication we have shown that 17α-hydroxylase and C17–20-lyase, enzymes involved in the formation of androgens (C19-steroids) from C21-steroids (progestins), are present in day-6 rabbit blastocysts. C17–20-lyase activity was undetectable to low in day-5 and increased in day-6 balstocysts. The activity was further increased in day-6 blastocysts cultured for 24 h. Because prostaglandins have been implicated in uterine vascular changes at about the time of implantation and pregnancy establishment, and because catechol estrogens are more potent than phenolic estrogens in stimulating prostaglandin synthesis in the blastocyst and the uterus, we determined catechol estrogen forming capacity in the rabbit and pig blastocyst. Catechol estrogen forming capacity (estrogen-2/4-hydroxylase) in the pig blastocyst appears on day 10 of pregnancy, peaks on day 12 and then declines. Our preliminary experiments also indicate that day-6 rabbit blastocysts have catechol estrogen forming capacity. On the basis of our present findings and of others, we propose that catechol estrogens of embryonic origin mediate the stimulatory effect of estrogens on prostaglandin synthesis in the embryo and/or the uterus and thus participate in the process of establishment of pregnancy.

Role of embryonic oestrogen in rabbit blastocyst development and metabolism.

Rabbit morulae were grown for 24 h in Ham's F12 medium supplemented with BSA. CI-628 citrate (1.5 micrograms/ml), a specific oestrogen antagonist, significantly inhibited the transformation of morulae to blastocysts. This inhibition was reversed with oestradiol-17 beta (1 micrograms/ml) but not oestradiol-17 alpha (1 micrograms/ml) added to the culture medium. The specific activities of phosphofructokinase, lactic dehydrogenase, malate dehydrogenase and alkaline phosphatase in blastocysts grown in vitro for 24 h in medium TC 199 + BSA showed significant elevation with blastocyst growth and expansion, while that of acid phosphatase revealed no change, and leucine aminopeptidase activity declined significantly. These changes were markedly inhibited by CI-628 citrate (2 micrograms/ml) and were reversed by oestradiol-17 beta (0.4 micrograms/ml) but not by oestradiol-17 alpha (0.4 micrograms/ml). Our findings suggest a role of oestrogen present in the rabbit morula and blastocyst in the triggering of embryonic differentiation and metabolic functions.

Chorionic gonadotropin receptors and immunoreactive chorionic gonadotropin in implantation of the rabbit blastocyst

To determine the temporal relationship between immunoreactive chorionic gonadotropin, chorionic gonadotropin receptors, and implantation of the rabbit blastocyst, (1) immunoreactive chorionic gonadotropin in plasma, uterine flushings and blastocysts; (2) chorionic gonadotropin receptors on blastocysts (day 5 and 6) and embryonic and interembryonic segments of the uterus (day 7); and (3) chorionic gonadotropin receptors on the endometrium and myometrium (day 1 through 6) were measured. Binding of I125-labeled β-subunit of human chorionic gonadotropin (hCG) by cell membranes from blastocysts increased significantly from 6.0 ± 1.1 fmol/mg of protein (mean ± SE) on day 5 (N = 6) to 16.1 ± 1.3 fmol/mg of protein on day 6 (n = 6) (P < 0.001). Immunoreactive chorionic gonadotropin levels in blastocyst fluid increased from 0.3 ng/ml of day 5 to 0.65 ng/ml on day 6. Specific binding of I125-labeled hCG was absent in endometrial and myometrial cell membranes before implantation (days 1 to 6) but was found in decidual cells from embryonic segments on day 7. Plasma immunoreactive chorionic gonadotropin or chorionic gonadotropin-like material increased from 6 ng/ml of day 1 to 52 ng/ml on day 7. Uterine flushings had chorionic gonadotropin levels of 0.4 ng/ml of day 2, which increased slowly to 1.0 ng/ml on day 7. Intrauterine instillation of hCG into nonpregnant uterine horns demonstrated transuterine absorption of hCG with plasma hCG levels showing a dose-related response. Our findings demonstrate that (1) immunoreactive chorionic gonadotropin or chorionic gonadotropin-like material is detectable in plasma, uterine flushings, and blastocyst fluid before implantation, (2) chorionic gonadotropin receptors are present on the blastocyst cells before implantation, and (3) the uterus can absorb chorionic gonadotropin from its lumen.

Different environmental stresses can activate the expression of a heat shock gene in rabbit blastocysts

AbstractWe have utilized the rabbit 6‐day blastocyst as a model system in which to examine the effect of environmental stress on embryonic gene expression. Elevation of the incubation temperature from 37 to 43 °C, exposure to 50 μM sodium arsenite or mechanical injury (resulting in the structural collapse of the 6–day rabbit blastocyst) was found to depress total protein synthesis as well as enhance the synthesis of a 70,000‐dalton stress‐induced protein. The molecular mass of this stress protein is similar to a heat shock protein (HSP) found in other eukaryotic systems. A recombinant DNA probe consisting of the 5′ end of a mouse gene for a 70,000‐dalton HSP hybridized to RNA isolated from heat shocked, sodium arsenite‐treated, and mechanically injured blastocysts but not to RNA isolated from control embryos. These results as well as in vitro translation data suggest that the expression of the 70 K HSP is controlled at the transcriptional level. The levels of actin mRNA, as detected by means of a recombinant DNA probe encoding a Drosophila actin gene, did not undergo a major alteration following these different stresses. The relevance of these observations to embryonic cellular homeostatis is discussed.

Ultrastructural observations on rabbit blastocysts after maternal exposure to cadmium chloride.

Cadmium chloride (CdCl2) (0.1 or 1.0%) was given in the drinking water to pregnant rabbits on Days 0 to 5 of gestation. Electron microscopy of the blastocysts revealed significant changes in the lysosomes (e.g. autophagic vacuoles and residual bodies) of endodermal cells as well as those of the inner cell mass. No such changes were evident in the trophoblast.

Indomethacin (1) inhibits implantation of transferred rabbit blastocysts by an endometrial effect

Indomethacin (1) inhibits implantation of transferred rabbit blastocysts by an endometrial effect

Variability in different lots of commercial bovine serum albumin affects cell multiplication and hatching of rabbit blastocysts in culture.

Rabbit morulae were cultured in vitro for 4 days in a synthetic culture medium supplemented with two different lots of commercial bovine serum albumin (BSA) and two different amino acid formulations in a factorial 2 x 2 arrangement. One lot of BSA caused complete hatching of a proportion of blastocysts and formation of more than twice as many cells per blastocyst (hatched and unhatched) as that of the second BSA lot which did not cause complete hatching of any blastocysts. The mean cell numbers of hatched blastocysts were more than twice those of non-hatched blastocysts. There was no significant effect of amino acid formulation.

Are blastocyst prostaglandins produced endogenously?

This study was undertaken to determine whether preimplantation mouse and rabbit blastocysts possess cyclooxygenase activity and therefore are able to metabolize arachidonic acid (AA) to prostaglandins (PGs) and thromboxane B2. Single rabbit blastocysts or groups of 100 mouse blastocysts were incubated for 4 h in the presence of 5 muCi/ml [3H] AA (sp. ac. 61 Ci/mmol) or for 24 h and 18 h with 0.2 and 0.1 muCi/ml [14C] AA (sp. ac. 56.5 Ci/mmol), respectively. Incubated blastocysts were subsequently exposed for 10 min to 24 h to the Ca2+ specific ionophore, X-537A (10 microM), to stimulate release of radiolabeled AA from the phospholipid pool. After incubation, incorporation of AA into the phospholipid and neutral lipid pools, and metabolism of the fatty acid to PGs and thromboxane B2, were determined using thin-layer chromatographic (TLC) and liquid scintillation spectroscopic techniques. In addition, spent incubation media were analyzed for radiolabeled PG content. In blastocysts of both species, incorporation of AA into phospholipids was greater than that into neutral lipids (mouse: 1.0 vs. 0.7 pmol/blastocyst; rabbit: 159.7 vs. 56.9 pmol/blastocyst). The ionophore stimulated the release of AA from the phospholipid, and to a lesser extent, from the neutral lipid pool, of both blastocysts. No newly synthesized PGs were detected in either mouse blastocysts or their spent incubation media after stimulation with X-537A. Radiolabeled PGs (PGE2, PGF2 alpha, PGD2, PGA2) and thromboxane B2 were present in the media of rabbit blastocyst incubations, however, but were undetectable in the tissue extracts. Increases in metabolism of AA into each compound were observed with an increase in time of exposure to ionophore, and meclofenamic acid (2 microM) partially inhibited the synthesis of all compounds during a 24-h incubation. The results are discussed with regard to the role of blastocyst PGs in the events of implantation.